Abnormal Ca-cycling, which is a universal characteristic of experimental and human heart failure, is partially due to impaired calcium sequestration into the sarcoplasmic reticulum (SR). SR calcium uptake is mediated by a Ca2+-ATPase (SERCA2), whose activity is reversibly regulated by phospholamban (PLN). Dephosphorylated phospholamban inhibits SERCA2 and phosphorylation relieves this inhibition. In human and experimental heart failure, phospholamban is highly dephosphorylated, due to increased SR protein phosphatase activity, resulting in higher inhibition of SERCA2 and cardiac deterioration. However, our initial simple view of a PLN/SERCA2 complex has been modified by our recent identification of a regulatome consisting of SERCA2, PLN, the regulatory subunit of protein phosphatase 1 (RGL), that anchors this enzyme to PLN, and the two regulators of protein phosphatase 1 (PP1): inhibitor-1 and the small heat shock protein 20 (Hsp20). Hsp20 physically interacts with PP1 and inhibits its activity, resulting in increased PLN phosphorylation and contractility. Hsp20 is also an anti-apoptotic protein protecting the heart against stress-induced injury. Our hypothesis is that Hsp20 is a fundamental regulator of calcium cycling and cell survival in the heart. Alterations in the levels or activity of Hsp20 will lead to disruption f these processes, impacting cardiac remodeling. Indeed, a human mutant of Hsp20 abrogates its stimulatory effects on Ca-cycling and cardioprotection. The innovation of this proposal is the firs identification of a Hsp20/PP1/RGL/PLN/SERCA2 interactome. Our goal is to define the triggers and mechanisms that disrupt the function of this SR regulatome in response to aging and clinically relevant stress with specific emphasis on the newly-discovered regulator, Hsp20. Hsp20 is also phosphorylated by the ?-adrenergic axis but the significance of this phosphorylation remains elusive. Accordingly, our Aims will provide a first comprehensive characterization of the: 1) molecular mechanisms by which Hsp20 regulates PP1 activity, the downstream substrates and its interaction with inhibitor-1; 2) functional role of PKA-phosphorylation of Hsp20 in regulation of contractility and cell survival, impacting the processes of aging and cardiac remodeling, following myocardial ischemia; 3) therapeutic potential of Hsp20 in inhibition of cardiac dysfunction and pathological growth; and 4) pathways underlying abrogation of the Hsp20 beneficial effects on contractility and cardioprotection elicited by the human S10F- Hsp20 mutant. We will employ an integrative approach using molecular, biochemical and physiological methodology. Our pilot studies are exciting and the proposed experiments are feasible and highly relevant, since the levels and activity of protein phosphatase 1 are significantly increased in failing hearts. The findings will provide fundamental insights into the mechanisms regulating the PP1/PLN/SERCA axis, remodeling and apoptosis with emphasis in the pathophysiology of heart failure and may reveal Hsp20 as a new target to enhance both contractility and cell survival in the stressed heart.